- Methodology article
- Open Access
Quantitative detection of free 24S-hydroxycholesterol, and 27-hydroxycholesterol from human serum
© Bandaru and Haughey; licensee BioMed Central. 2014
- Received: 20 June 2014
- Accepted: 17 December 2014
- Published: 24 December 2014
Cholesterol metabolism is important for the maintenance of myelin and neuronal membranes in the central nervous system. Blood concentrations of the brain specific cholesterol metabolite 24S-hydroxysterol to the peripheral metabolite 27-hydroxycholesterol may be useful surrogate markers for neurodegenerative diseases including Alzheimer’s disease, Huntington’s disease, HIV-Associated Neurocognitive Disorders, and Multiple Sclerosis. However, current methods to isolate hydroxycholesterols are labor intensive, prone to produce variable extraction efficiencies and do not discriminate between free and esterfied forms of hydroxycholesterols. Since free hydroxycholesterols are the biologically active form of these sterols, separating free from esterfied forms may provide a sensitive measure to identify disease-associated differences in brain sterol metabolism.
We found that average human serum concentrations were 12.3 ± 4.79 ng/ml for free 24(s)-hydroxycholesterol and 17.7 ± 8.5 ng/ml for 27-hydroxycholesterol.
Serum measurements of these biologically active oxysterols may be useful surrogate measures for brain health in a variety of neurodegenerative conditions.
- Multiple sclerosis
- HIV associated neurocogntiive disorder
Hydroxycholesterols are mono-oxygenated derivatives of cholesterol (cholesten-5-3β-ol) that comprise a family of polycyclic compounds that contain a second oxygen atom as a hydroxyl group on the skeleton of cholesterol. In vivo, hydroxycholesterols are present as unesterified (biologically active) and esterified forms (largely biologically inert)  Hydroxycholesterols are formed through enzymatic conversion of cholesterol or by free radical autoxidation, and exhibit a short half life relative to cholesterol. Hydroxycholesterols are important intermediates in a number of catabolic pathways that regulate a variety of biological effects. For example, hydroxycholesterols are important for cholesterol transport from the periphery to the liver , modulate the expression of sterol sensitive genes involved in lipid and sterol biosynthesis ,, act as substrates for the formation of bile salts , serve as ligands that activate nuclear liver X receptors-α and -β , and are involved in the regulation of cholesterol and lipid metabolism and homeostasis ,. In the central nervous system, hydroxycholesterols regulate arachidonic acid release, voltage-gated calcium channels, synaptic plasticity, induce IL-8, promote neurogenesis and induce apoptosis -. Consistent with these important roles for regulating biological functions, levels of free hydroxycholesterols are extremely low and tightly controlled, with the majority of hydroxycholesterols maintained in esterified forms ,.
Many cell types have the ability to oxygenate cholesterol by mechanisms that involve the cytochrome P450 family of oxidases (CYP). Cell type specific expression CYP subtypes results in the tissue-specific production of particular oxysterol species. Several CYP are present in the central nervous system including 24S-hydroxycholesterolhydroxylase (CYP46), a P450 family member that is expressed in neurons, glia and in endothelial cells of the blood–brain barrier . 24S-hydroxycholesterol (Cholest-5-en-3β, 24(S)-diol) is the most abundant hydroxycholesterol in brain and is the primary transport form of cholesterol from the central nervous system into the blood, with smaller amounts eliminated through cerebrospinal fluid . It has been suggested by several studies that serum or plasma levels of 24S-hydroxycholesterol may reflect brain developmental and neuropathological changes associated with Alzheimer’s disease (AD), Huntington’s disease and Multiple Sclerosis -. 24S-hydroxycholesterol is often expressed as a ratio to 27-hydroxycholesterol (25R-Cholest-5-en-3β, 26-diol). 27-hydroxycholesterol is formed primarily in the periphery by the P450 enzyme sterol 27-hydroxylase (CYP27) . CYP27 is expressed in arterial endothelium, macrophages and to lesser extents in other tissues such as cortex, spleen, liver, kidney, adrenal gland and heart ,. 27-hydroxycholesterol can function as a ligand for nuclear receptors, liver X receptors (LXR) and farnesoid X-activated receptors (FXRs) . 27-hydroxycholesterol can also regulate hydrocymethylglutaryl-CoA reductase ,, and enhances cholesterol efflux from the vascular endothelium . Macrophages have the highest capacity to convert cholesterol to 27-hydroxycholesterol, which is then transported in blood to the liver where it is converted to bile acids .
Hydroxycholesterol detection and quantification has been accomplished in a variety of tissues by isotope-dilution gas-chromatography-mass spectrometry (GC-MS) ,, gas- and high-performance liquid chromatography (HPLC)/mass spectrometry , HPLC with UV detection of cholesterol oxidation products in tissues , as ∆4- 3-ketone derivatives by HPLC  and as derivatives of GP hydrazones . Because “free” hydroxycholesterol levels are below the detection limits of many instruments, saponification and/or solid phase extraction techniques have typically been used to extract “total” hydroxycholesterols. The primary advantage of these methods is a high yield of hydroxycholesterols. The disadvantages include lengthy sample preparation times, sample loss, inconsistent yields, and the inability to discriminate between free and esterified hydroxycholesterols. In this study we developed a simple and direct extraction protocol and sensitive LC/ESI/MS/MS method for separation and simultaneous quantitative determination of free 24S-hydroxycholesterol and 27-hydroxycholesterol in serum.
Identification and Optimization of ESI/MS/MS for 24S-hydroxycholesterol and 27-hydroxycholesterol
Molecular and fragment ion m/z, and associated parameters for detection and quantification of 24S-hydroxycholesterol, 27-hydroxycholesterol and 24(R/S) hydroxycholesterol (d6) individual species by MRM
24(R/S) hydroxycholesterol (d6)
Optimization of HPLC conditions for separation of hydroxycholesterols
Recovery, accuracy and precision
Precision and accuracy of hydroxycholesterols
a. Inter-day analysis
b. Intra-day analysis
Linearity, limits of detection and quantification
Linearity calculated by regression analysis from standard curves of hydroxycholesterols
Correlation coefficient (R)
The goal of this study was to develop an efficient and rapid extraction protocol for LC/ESI/MS/MS-based detection and quantification of free 24S-hydroxycholesterol and 27-hydroxycholesterol from human serum. For clinical studies, and for the potential use of these hydroxysterols as surrogate markers, it is important that sample analysis be rapid and cost effective. Here we present a simple, inexpensive, and rapid protocol for the extraction of 24S-hydroxycholesterol and 27-hydroxycholesterol from human serum. In addition, the simplicity of the extraction method increases data reproducibility by decreasing variability of product yield. This low cost and rapid sample preparation coupled with the high sensitivity of LC/MS/MS instruments and accurate quantification by MRM make this a potentially powerful approach for the high-throughput quantification of hydroxycholesterol species in clinical and experimental samples.
This extraction method does not use saponification and therefore measures free hydroxycholesterols. This is an important consideration since free hydroxycholesterols are the most biologically active form of these sterols ,. In our healthy human volunteers, serum concentrations ranged from 4 to 21 ng/ml for 24S-hydroxycholesterol and 4 to 29 ng/ml for 27-hydroxycholesterol. These ranges are considerably lower than previously reported levels of total hydroxycholesterol in human serum that range from 60 to 83 ng/ml for 24S-hydroxycholesterol and 120 to 159 ng/ml for 27-hydroxycholesterol ,,,. These data are consistent with findings that suggest more than 80% of 24S-hydroxycholesterol and 27-hydroxychoelsterol are maintained in an esterfied state .
Since this is the first report of this extraction method, it is not possible to determine if measuring free hydroxycholesterols has a diagnostic or experimental advantage over measuring total hydroxycholesterols. In this study we analyzed a small number of samples to validate the method and did not compare to a disease state. However, we have recently used this method to quantitatively measure levels of free 24S-hydroxycholesterol and 27-hydroxycholesterol in serum of subjects who later developed cognitive impairment, and found that increased levels of free 24S-hydroxycholesterol and the 24S-hydroxycholesterol/total cholesterol ratio were associated with greater risk of impairment on tasks that assess psychomotor speed and executive functioning, while higher levels of free 27-hydroxycholesterol and the 27-hydroxycholesterol/total cholesterol ratio were associated with greater risk of delayed memory impairment. These data were qualitatively different from previous reports that measured total serum levels of these hydroxysterols. For example, total 27-hydroxycholesterol to total cholesterol ratio was associated with a faster decline of immediate memory recall over six years of follow-up . Although, a second reported study did not find an association between total serum 24-hydroxycholesterol or total 27-hydroxycholesterol and cognitive performance , this study measured total hydroxysterols. To date, only a single study has directly compared free to total levels of 24S-hydroxycholesterol and 27-hydroxycholesterol in serum. In this study it was reported that 80% of 24S-hydroxycholesterol and 85% of 27-hydroxycholesterol is esterfied in healthy volunteers. Males had higher levels of total 27-hydroxycholesterol compared to females. They found no other demographic or age-related differences in total 24S-hydroxycholesterol or 27-hydroxychoelsterol, and did not determine if there were age- or disease-related differences in free vs. esterfied hydroxycholesterols. Since it is the free forms of these hydroxysterols that have biological activity, and free forms are less than 20% of total hydroxysterols, it is possible that measuring total 24S-hydroxycholesterol and 27-hydroxycholesterol could mask a biological or disease-associated effect. These findings suggest that there may be important differences in free vs. total 24S-hydroxycholesterol and 27-hydroxycholesterols in relation to sex, age and neurodegenerative conditions that merit further study.
The ability to economically and efficiently measure 24S-hydroxycholesterol and 27-hydroxycholesterol in serum may also be useful as surrogate measures for the effectiveness of chemotherapeutics. The approximate cost per sample for this rapid extraction method is $3.00. Saponification with solid phase extraction increases the approximate cost $18.00/sample. These costs are for sample processing and do no include mass spectrometry time that is equal for both methods. Likewise sample run times are 12 min/sample regardless of the extraction method. A number of sterol modifying agents are being tested as potential therapeutics for neurodegenerative disease -, and it is possible that serum measures of 24S-hydroxycholesterol and 27-hydroxycholesterol may have utility as rapid and inexpensive surrogate markers to efficiently determine the effectiveness of therapeutics.
Serum measurements of these biologically active hydroxycholesterols may be useful surrogate measures for brain health in a variety of neurodegenerative conditions.
Chemicals and equipment
All solvents and chemicals were HPLC grade. Methanol (CH3OH), ethanol (C2H5OH) and diethyl ether (CH3-CH2-O-CH2-CH3) were purchased from Fisher Scientific (USA), ammonium formate (HCOONH4) and formic acid (HCOOH) were purchased from Sigma Aldrich (St. Louis, MO). 24S-hydroxycholesterol and 27-hydroxycholesterol were purchased form Research Plus Inc. (Barnegat, NJ). Internal standard 24(R/S)-hydroxycholesterol (d6) [cholest-5-ene-3β, 24(R/S)-diol (d6)] was purchased from Avanti Polar Lipids (Alabaster, AL). Glass vials were purchased from Agilent Technologies, Inc. (Santa Clara, CA). Isotemp vacuum oven was purchased from Fisher Scientific (Model 285A, Pittsburgh, PA). Borosilicate-coated glass tubes and pipettes were used to reduce adhesion of sterols to plastic and glassware (Fisher Scientific, Pittsburgh, PA).
Human serum was obtained from 12 healthy volunteers (5 males and 7 females) age 25–36 years at the Johns Hopkins University School of Medicine. Approximately 8 ml (human) blood was collected into BD P100 tubes Sodium heparin (Beckton Dickenson, Franklin Lakes, NJ). Tubes were inverted 8–10 times to mix the protease inhibitors and anticoagulent with the blood sample then placed onto ice. Blood was then centrifuged at 2000 g at 4°C for 15 min. Serum was aliquoted and transferred into cryovials for storage at −80°C until use. All samples underwent a single freeze-thaw cycle before use.
Extraction of 24S-hydroxycholesterol and 27-hydroxycholesterol
We developed a single step direct extraction method for both 24S-hydroxycholesterol and 27-hydroxycholesterol. For extraction, 0.5 ml of Serum was transferred into a glass tube and 5 μl of 24(R/S)-hydroxycholesterol (d6) (internal standard) from 100 μg/ml stock was added, followed by 3 ml of pure ethanol and the mixture vortexed. Diethyl ether (4 ml) was then were added and the mixture vortexed and centrifuged at 4,000 g for 10 minutes. The supernatant was separated and the residue was re-extracted using the same volumes of solvents as was used in the initial extraction. Supernatants were mixed together, and dried under a stream of nitrogen or in a vacuum oven at 30°C (we did not observe any qualitative or quantitative differences when samples were dried under nitrogen compared with a vacuum oven). Dried extracts were re-suspended into 100 μl of methanol, vortexed, centrifuged and transferred to an autosampler vial insert where it was maintained at 4°C. Samples were injected into the HPLC using an Agilent 1100 series autosampler (Agilent Technologies, Inc., Santa Clara CA, United States).
Quantification of hydroxycholesterols by LC/ESI/MS/MS using multiple reaction monitoring (MRM)
Gradient conditions for LC
Total time (min)
Flow rate (μl/min)
Mobile phase A* (%)
Mobile phase B* (%)
Standards for quantitative analysis and correction of extraction efficiency
Stock solutions for 24S-hydroxycholesterol, 27-hydroxycholesterol, and 24(R/S)-hydroxycholesterol (d6) (internal standard) were prepared separately in CH3OH to produce a concentration range of 10 ng/ml to 1000 ng/ml. Samples were spiked with 1 μg/ml 24(R/S)-hydroxycholesterol (d6) that was used as an internal control to correct for slight differences in ionization efficiency, chromatographic retention, fragmentation and molecular interactions between hydroxycholesterol species. Blank serum samples were used for background correction. Calibration curves were plotted using the peak area ratios of 24S-hydroxycholesterol or 27-hydroxycholesterol to 24(R/S)-hydroxycholesterol (d6). All stock solutions were stored at −20°C. Calibrations were conducted using least squares linear regression.
Standard protocol approvals, registrations, and patient consents
The collection and use of human samples was approved by the IRB the Johns Hopkins University SOM and included written and informed consent for the use of Serum for research purposes.
This research was supported by the National Institute of Health grants AG023471, MH077542 and AA017408 to NJH. The authors would like to thank Fang Yang and Lepakshi Sahni for expert technical assistance.
- Brown AJ, Jessup W: Oxysterols and atherosclerosis. Atherosclerosis. 1999, 142 (1): 1-28. 10.1016/S0021-9150(98)00196-8.View ArticlePubMedGoogle Scholar
- Bjorkhem I, Andersson O, Diczfalusy U, Sevastik B, Xiu RJ, Duan C, Lund E: Atherosclerosis and sterol 27-hydroxylase: evidence for a role of this enzyme in elimination of cholesterol from human macrophages. Proc Natl Acad Sci U S A. 1994, 91 (18): 8592-8596. 10.1073/pnas.91.18.8592.PubMed CentralView ArticlePubMedGoogle Scholar
- Brown MS, Goldstein JL: Suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and inhibition of growth of human fibroblasts by 7-ketocholesterol. J Biol Chem. 1974, 249 (22): 7306-7314.PubMedGoogle Scholar
- Kandutsch AA, Chen HW, Shown EP: Binding of 25-hydroxycholesterol and cholesterol to different cytoplasmic proteins. Proc Natl Acad Sci U S A. 1977, 74 (6): 2500-2503. 10.1073/pnas.74.6.2500.PubMed CentralView ArticlePubMedGoogle Scholar
- Danielsson H: Present Status of Research on Catabolism and Excretion of Cholesterol. Adv Lipid Res. 1963, 1: 335-385. 10.1016/B978-1-4831-9937-5.50015-6.PubMedGoogle Scholar
- Bjorkhem I, Meaney S, Diczfalusy U: Oxysterols in human circulation: which role do they have?. Curr Opin Lipidol. 2002, 13 (3): 247-253. 10.1097/00041433-200206000-00003.View ArticlePubMedGoogle Scholar
- Repa JJ, Mangelsdorf DJ: The role of orphan nuclear receptors in the regulation of cholesterol homeostasis. Annu Rev Cell Dev Biol. 2000, 16: 459-481. 10.1146/annurev.cellbio.16.1.459.View ArticlePubMedGoogle Scholar
- Schroepfer GJ: Oxysterols: modulators of cholesterol metabolism and other processes. Physiol Rev. 2000, 80 (1): 361-554.PubMedGoogle Scholar
- Ares MP, Porn-Ares MI, Thyberg J, Juntti-Berggren L, Berggren PO, Diczfalusy U, Kallin B, Bjorkhem I, Orrenius S, Nilsson J: Ca2+ channel blockers verapamil and nifedipine inhibit apoptosis induced by 25-hydroxycholesterol in human aortic smooth muscle cells. J Lipid Res. 1997, 38 (10): 2049-2061.PubMedGoogle Scholar
- Gabbi C, Kim HJ, Hultenby K, Bouton D, Toresson G, Warner M, Gustafsson JA: Pancreatic exocrine insufficiency in LXRbeta−/− mice is associated with a reduction in aquaporin-1 expression. Proc Natl Acad Sci U S A. 2008, 105 (39): 15052-15057. 10.1073/pnas.0808097105.PubMed CentralView ArticlePubMedGoogle Scholar
- Kolsch H, Ludwig M, Lutjohann D, Rao ML: Neurotoxicity of 24-hydroxycholesterol, an important cholesterol elimination product of the brain, may be prevented by vitamin E and estradiol-17beta. J Neural Transm. 2001, 108 (4): 475-488. 10.1007/s007020170068.View ArticlePubMedGoogle Scholar
- Kotti T, Head DD, McKenna CE, Russell DW: Biphasic requirement for geranylgeraniol in hippocampal long-term potentiation. Proc Natl Acad Sci U S A. 2008, 105 (32): 11394-11399. 10.1073/pnas.0805556105.PubMed CentralView ArticlePubMedGoogle Scholar
- Lahoua Z, Astruc ME, Barjon JN, Michel F, de Crastes Paulet A: Mechanism of the activation of arachidonic acid release by oxysterols in NRK 49 F cells: role of calcium. Cell Signal. 1989, 1 (6): 569-576. 10.1016/0898-6568(89)90065-X.View ArticlePubMedGoogle Scholar
- Lee CS, Park WJ, Han ES, Bang H: Differential modulation of 7-ketocholesterol toxicity against PC12 cells by calmodulin antagonists and Ca2+ channel blockers. Neurochem Res. 2007, 32 (1): 87-98. 10.1007/s11064-006-9230-8.View ArticlePubMedGoogle Scholar
- Sacchetti P, Sousa KM, Hall AC, Liste I, Steffensen KR, Theofilopoulos S, Parish CL, Hazenberg C, Richter LA, Hovatta O, Gustafsson JA, Arenas E: Liver X receptors and oxysterols promote ventral midbrain neurogenesis in vivo and in human embryonic stem cells. Cell Stem Cell. 2009, 5 (4): 409-419. 10.1016/j.stem.2009.08.019.View ArticlePubMedGoogle Scholar
- Trousson A, Bernard S, Petit PX, Liere P, Pianos A, El Hadri K, Lobaccaro JM, Ghandour MS, Raymondjean M, Schumacher M, Massaad C: 25-hydroxycholesterol provokes oligodendrocyte cell line apoptosis and stimulates the secreted phospholipase A2 type IIA via LXR beta and PXR. J Neurochem. 2009, 109 (4): 945-958. 10.1111/j.1471-4159.2009.06009.x.View ArticlePubMedGoogle Scholar
- Burkard I, von Eckardstein A, Waeber G, Vollenweider P, Rentsch KM: Lipoprotein distribution and biological variation of 24S- and 27-hydroxycholesterol in healthy volunteers. Atherosclerosis. 2007, 194 (1): 71-78. 10.1016/j.atherosclerosis.2006.09.026.View ArticlePubMedGoogle Scholar
- Dzeletovic S, Breuer O, Lund E, Diczfalusy U: Determination of cholesterol oxidation products in human plasma by isotope dilution-mass spectrometry. Anal Biochem. 1995, 225 (1): 73-80. 10.1006/abio.1995.1110.View ArticlePubMedGoogle Scholar
- Ramirez DM, Andersson S, Russell DW: Neuronal expression and subcellular localization of cholesterol 24-hydroxylase in the mouse brain. J Comp Neurol. 2008, 507 (5): 1676-1693. 10.1002/cne.21605.PubMed CentralView ArticlePubMedGoogle Scholar
- Heverin M, Meaney S, Lutjohann D, Diczfalusy U, Wahren J, Bjorkhem I: Crossing the barrier: net flux of 27-hydroxycholesterol into the human brain. J Lipid Res. 2005, 46 (5): 1047-1052. 10.1194/jlr.M500024-JLR200.View ArticlePubMedGoogle Scholar
- Koschack J, Lutjohann D, Schmidt-Samoa C, Irle E: Serum 24S-hydroxycholesterol and hippocampal size in middle-aged normal individuals. Neurobiol Aging. 2009, 30 (6): 898-902. 10.1016/j.neurobiolaging.2007.10.010.View ArticlePubMedGoogle Scholar
- Leoni V, Mariotti C, Tabrizi SJ, Valenza M, Wild EJ, Henley SM, Hobbs NZ, Mandelli ML, Grisoli M, Bjorkhem I, Cattaneo E, Di Donato S: Plasma 24S-hydroxycholesterol and caudate MRI in pre-manifest and early Huntington’s disease. Brain. 2008, 131 (Pt 11): 2851-2859. 10.1093/brain/awn212.View ArticlePubMedGoogle Scholar
- Lutjohann D, Bjorkhem I, Locatelli S, Dame C, Schmolling J, von Bergmann K, Fahnenstich H: Cholesterol dynamics in the foetal and neonatal brain as reflected by circulatory levels of 24S-hydroxycholesterol. Acta Paediatr. 2001, 90 (6): 652-657. 10.1080/080352501750258720.View ArticlePubMedGoogle Scholar
- Lutjohann D, Papassotiropoulos A, Bjorkhem I, Locatelli S, Bagli M, Oehring RD, Schlegel U, Jessen F, Rao ML, von Bergmann K, Heun R: Plasma 24S-hydroxycholesterol (cerebrosterol) is increased in Alzheimer and vascular demented patients. J Lipid Res. 2000, 41 (2): 195-198.PubMedGoogle Scholar
- Meng LJ, Griffiths WJ, Nazer H, Yang Y, Sjovall J: High levels of (24S)-24-hydroxycholesterol 3-sulfate, 24-glucuronide in the serum and urine of children with severe cholestatic liver disease. J Lipid Res. 1997, 38 (5): 926-934.PubMedGoogle Scholar
- Papassotiropoulos A, Lutjohann D, Bagli M, Locatelli S, Jessen F, Buschfort R, Ptok U, Bjorkhem I, von Bergmann K, Heun R: 24S-hydroxycholesterol in cerebrospinal fluid is elevated in early stages of dementia. J Psychiatr Res. 2002, 36 (1): 27-32. 10.1016/S0022-3956(01)00050-4.View ArticlePubMedGoogle Scholar
- Teunissen CE, Floris S, Sonke M, Dijkstra CD, De Vries HE, Lutjohann D: 24S-hydroxycholesterol in relation to disease manifestations of acute experimental autoimmune encephalomyelitis. J Neurosci Res. 2007, 85 (7): 1499-1505. 10.1002/jnr.21266.View ArticlePubMedGoogle Scholar
- Andersson S, Davis DL, Dahlback H, Jornvall H, Russell DW: Cloning, structure, and expression of the mitochondrial cytochrome P-450 sterol 26-hydroxylase, a bile acid biosynthetic enzyme. J Biol Chem. 1989, 264 (14): 8222-8229.PubMedGoogle Scholar
- Cali JJ, Russell DW: Characterization of human sterol 27-hydroxylase. A mitochondrial cytochrome P-450 that catalyzes multiple oxidation reaction in bile acid biosynthesis. J Biol Chem. 1991, 266 (12): 7774-7778.PubMedGoogle Scholar
- Javitt NB: 25R,26-Hydroxycholesterol revisited: synthesis, metabolism, and biologic roles. J Lipid Res. 2002, 43 (5): 665-670.PubMedGoogle Scholar
- Esterman AL, Baum H, Javitt NB, Darlington GJ: 26-hydroxycholesterol: regulation of hydroxymethylglutaryl-CoA reductase activity in Chinese hamster ovary cell culture. J Lipid Res. 1983, 24 (10): 1304-1309.PubMedGoogle Scholar
- Javitt NB: Bile acid synthesis from cholesterol: regulatory and auxiliary pathways. Faseb J. 1994, 8 (15): 1308-1311.PubMedGoogle Scholar
- Reiss AB, Martin KO, Javitt NB, Martin DW, Grossi EA, Galloway AC: Sterol 27-hydroxylase: high levels of activity in vascular endothelium. J Lipid Res. 1994, 35 (6): 1026-1030.PubMedGoogle Scholar
- Russell DW: Oxysterol biosynthetic enzymes. Biochim Biophys Acta. 2000, 1529 (1–3): 126-135. 10.1016/S1388-1981(00)00142-6.View ArticlePubMedGoogle Scholar
- Breuer O, Bjorkhem I: Simultaneous quantification of several cholesterol autoxidation and monohydroxylation products by isotope-dilution mass spectrometry. Steroids. 1990, 55 (4): 185-192. 10.1016/0039-128X(90)90109-O.View ArticlePubMedGoogle Scholar
- Sevanian A, Seraglia R, Traldi P, Rossato P, Ursini F, Hodis H: Analysis of plasma cholesterol oxidation products using gas- and high-performance liquid chromatography/mass spectrometry. Free Radic Biol Med. 1994, 17 (5): 397-409. 10.1016/0891-5849(94)90166-X.View ArticlePubMedGoogle Scholar
- Csallany AS, Kindom SE, Addis PB, Lee JH: HPLC method for quantitation of cholesterol and four of its major oxidation products in muscle and liver tissues. Lipids. 1989, 24 (7): 645-651. 10.1007/BF02535082.View ArticlePubMedGoogle Scholar
- Zhang Z, Li D, Blanchard DE, Lear SR, Erickson SK, Spencer TA: Key regulatory oxysterols in liver: analysis as delta4-3-ketone derivatives by HPLC and response to physiological perturbations. J Lipid Res. 2001, 42 (4): 649-658.PubMedGoogle Scholar
- Griffiths WJ, Wang Y, Alvelius G, Liu S, Bodin K, Sjovall J: Analysis of oxysterols by electrospray tandem mass spectrometry. J Am Soc Mass Spectrom. 2006, 17 (3): 341-362. 10.1016/j.jasms.2005.10.012.View ArticlePubMedGoogle Scholar
- Meaney S, Hassan M, Sakinis A, Lutjohann D, von Bergmann K, Wennmalm A, Diczfalusy U, Bjorkhem I: Evidence that the major oxysterols in human circulation originate from distinct pools of cholesterol: a stable isotope study. J Lipid Res. 2001, 42 (1): 70-78.PubMedGoogle Scholar
- Meaney S, Lutjohann D, Diczfalusy U, Bjorkhem I: Formation of oxysterols from different pools of cholesterol as studied by stable isotope technique: cerebral origin of most circulating 24S-hydroxycholesterol in rats, but not in mice. Biochim Biophys Acta. 2000, 1486 (2–3): 293-298. 10.1016/S1388-1981(00)00070-6.View ArticlePubMedGoogle Scholar
- Babiker A, and Diczfalusy: Transport of side-chain oxidized oxysterols in the human circulation. Biochem Biophysics Acta 1998(1392):333–339.Google Scholar
- Burkard I, Rentsch KM, von Eckardstein A: Determination of 24S- and 27-hydroxycholesterol in plasma by high-performance liquid chromatography-mass spectrometry. J Lipid Res. 2004, 45 (4): 776-781. 10.1194/jlr.D300036-JLR200.View ArticlePubMedGoogle Scholar
- van den Kommer TN, Dik MG, Comijs HC, Fassbender K, Lutjohann D, Jonker C: Total cholesterol and oxysterols: early markers for cognitive decline in elderly?. Neurobiol Aging. 2009, 30 (4): 534-545. 10.1016/j.neurobiolaging.2007.08.005.View ArticlePubMedGoogle Scholar
- Teunissen CE, De Vente J, von Bergmann K, Bosma H, van Boxtel MP, De Bruijn C, Jolles J, Steinbusch HW, Lutjohann D: Serum cholesterol, precursors and metabolites and cognitive performance in an aging population. Neurobiol Aging. 2003, 24 (1): 147-155. 10.1016/S0197-4580(02)00061-1.View ArticlePubMedGoogle Scholar
- Locatelli S, Lutjohann D, Schmidt HH, Otto C, Beisiegel U, von Bergmann K: Reduction of plasma 24S-hydroxycholesterol (cerebrosterol) levels using high-dosage simvastatin in patients with hypercholesterolemia: evidence that simvastatin affects cholesterol metabolism in the human brain. Arch Neurol. 2002, 59 (2): 213-216. 10.1001/archneur.59.2.213.View ArticlePubMedGoogle Scholar
- Davis W: The cholesterol transport inhibitor U18666a regulates amyloid precursor protein metabolism and trafficking in N2aAPP “Swedish” cells. Current Alzheimer Res. 2008, 5 (5): 448-456. 10.2174/156720508785908900.View ArticleGoogle Scholar
- Davis HR, Murgolo NJ, Graziano MP: Simvastatin with or without ezetimibe in familial hypercholesterolemia. N Engl J Med. 2008, 359 (5): 531-author reply 532–533PubMedGoogle Scholar
- Whitfield JF: The road to LOAD: late-onset Alzheimer’s disease and a possible way to block it. Expert Opin Ther Targets. 2007, 11 (10): 1257-1260. 10.1517/1472818.104.22.1687.View ArticlePubMedGoogle Scholar
- Vaya J, Schipper HM: Oxysterols, cholesterol homeostasis, and Alzheimer disease. J Neurochem. 2007, 102 (6): 1727-1737. 10.1111/j.1471-4159.2007.04689.x.View ArticlePubMedGoogle Scholar
- Papassotiropoulos A, Wollmer MA, Tsolaki M, Brunner F, Molyva D, Lutjohann D, Nitsch RM, Hock C: A cluster of cholesterol-related genes confers susceptibility for Alzheimer’s disease. J Clin Psychiatry. 2005, 66 (7): 940-947. 10.4088/JCP.v66n0720.View ArticlePubMedGoogle Scholar
- Chang TY, Chang CC, Bryleva E, Rogers MA, Murphy SR: Neuronal cholesterol esterification by ACAT1 in Alzheimer’s disease. IUBMB Life.Google Scholar
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